On the basis of our thermal denaturation studies with human albumin, we predict that the ability of ligands to induce biphasic (or multiphasic) protein denaturation should be independent of the means used to effect protein denaturation. Thus, recently we began an investigation of the effect of ligands on the guanidine HC1-induced unfolding of human albumin. We anticipate that biphasic denaturation should occur in the presence of subsaturating levels of a high affinity ligand. A preliminary experiment with undefatted human albumin, which contained - 1.4 mol of bound high-affinity, endogenous, long-chain fatty acid/ mol of albumin monomer and which corresponds to -15% saturation, showed biphasic denaturation when monitored by UV protein difference spectroscopy and by changes in intrinsic protein fluorescence. We plan to look at the effect of ligands of low, intermediate, and high affinity on the guanidine HC1 induced denaturation of defatted human albumin using UV difference spectroscopy, change in intrinsic protein fluorescence, and if possible, changes in circular dichroism.